The process of discovering a new therapeutic traditionally involves the following stages: (1) identification of a drug target, (2) validation of the target, (3) screening for compounds that affect the activity of the target, (4) testing lead compounds for toxicity, (5) testing lead compounds for side effects, and (6) examining the metabolism and stability of lead compounds, in the patient or in an appropriate model system.
High throughput screening (HTS) is one of the initial stages of the drug discovery process. It allows for testing of hundreds of thousands of chemical compounds per day to select the most prominent candidates for future examination. The compounds are tested against therapeutics targets. Recent developments in modern mass screening are highly influenced by the increasing number of targets identified by genomics and by the expansion of the libraries of compounds synthesized using methods of combinatorial chemistry.
For example, the plasma membrane plays host to more than 20 different families of receptors, including over 1000 different proteins, which have dubbed the receptorome. The G-protein coupled receptor (GPCR) superfamily represents the single largest slice of receptorome, although receptorome also includes toll-like receptors, integrin receptors, low-density lipoprotein receptors, protein tyrosine kinases receptors and phosphatases, cytokine receptors and even some ion channels that function as receptors.
The therapeutic exploitation of the interaction between extracellular and cell surface receptors, which originated as the “drug-receptor” concept, is considered to be one of the great ideas and insights in 20th-century biomedical science. Because of continuing advances in target identification, screening technologies and target validation, receptorome-based drug discovery efforts are likely to be productive for many decades to come. Not surprisingly, most experts conclude that the receptorome accounts for the largest portion in the druggable genome, with GPCR consistently leading the pack.
One of the first technologies for massive screening is the competition radioligand binding assay that relies on the use of high specific activity radioligands that selectively target the receptor of interest. Competition radioligand binding assays, as typically carried out, provide a reliable estimate of drug affinities for particular molecular targets but do not give information related to efficacy (as either agonist, antagonists or partial agonists). Traditionally, pharmacologists have relied on competition radioligand binding assays to measure ligand affinities and receptor specificities, as well as ascribe physiological relevance to GPCR. Competition radioligand screens are amenable to near—HTS techniques because they can be performed in 96+ well plates, which have been proven invaluable for the efficient screening of focused chemical libraries against an array of receptors. Competition radioligand binding assays are also helpful to link chemical structure with drugs side effects.
Even though radioligand screens are consistent across different cellular expression systems they have several drawbacks that have fueled research on alternative technologies. For example, radioligand assays do not differentiate between agonists, partial agonists, antagonists and inverse agonists. But more importantly, radioligand assays fail to detect responses that occur downstream of ligand binding and as such are not suited to deorphanizing orphan receptors because, by definition, these oGPCR have unknown ligands. Additionally, radioligand-binding assays are, typically, biased for detecting ligand binding to the endogenous receptor site (orthosteric site) and therefore might not detect small molecule modulators that exert their effect at sites distinct form the endogenous site, called allosteric site.
In contrast to radioligand binding assays, functional assays produce information rich ligand profiles that reveal how ligand modulate signal transduction for example in GPCR. Such functional assays rely on the detection of second messengers, which are produced as a result of receptor-specific signal transduction pathways. One of such methods, use the intracellular rise of calcium measured with a calcium sensitive fluorophore as signal while other methods use calcium or cAMP sensitive promoters coupled to reporters like luciferase to measure receptor activation or inhibition. The intracellular rise in calcium is measured with a calcium sensitive dye that increases its fluorescence as intracellular dye binds calcium or by a calcium sensing protein called aequorin that generates a luminescent signal when a coelenterazine derivative is added. But both calcium assays have the following disadvantages: (1) they can not be used to screen for inverse agonist; (2) the short time interval between ligand addition and calcium rise demands highly specialized equipment for simultaneous ligand addition and calcium measurement; and (3) the signal is not amplified. There are many technologies that measure cell or membrane based cAMP accumulation such as SPA™ (GE Healthcare), FlashPlate™ (Perkin Elmer), AlphaScreen™ (Perkin Elmer), HTRF cAMP (Cisbio) and HitHunter™ (DiscoveRx). Reporter gene-based screening technologies are cell-based assays where the increase in second messengers induces the expression of reporter molecules for example luciferase, beta-lactamase, SEAP and beta-galactosidase.
An ideal screening technology should be simple, nonradioactive, with high signal-to-noise ratio, homogeneous, with minimal reagents additions and be amenable to a microtiter plate format to facilitate robotic automation. Another consideration is whether to measure a proximal or distal signaling step. Measurement of events proximal to target activation will reduce the incidence of false positives; however signal-to-noise ratios can be enhanced moving down the signal transduction cascade owing to signal amplification. Another drawback of the use of reporter molecules coupled to second messengers like calcium or cAMP sensitive promoter is that those assays rely on inducible promoters that are usually weak promoters with a high background and that the reporter needs to be measured after transcription and translation either in lysates or as secreted products. The use of methods in which reporter molecules are rapidly secreted to the extracellular medium upon ligand-protein interaction would be desirable in drug discovery screening because it eliminates the cell lysis step to release the intracellular reporter. Also, the use of methods like the measurement of intracellular calcium with specific fluorophores eliminates the need of transcription and new protein synthesis, thus reducing the assay times. This reduction in assay time is very important in methods like homogeneous 3456 nanoplate screening, where the reaction volume is very low thus making reagents evaporation especially relevant. Also, the use of nanoplates for screening demands very sensitive methods for quantifying very small quantities of reporter molecules or second messengers and thus reporter molecules which can be coupled at will with signal amplification cascades are desirable. But as background is also amplified in signal amplification cascades, especially background due to the first steps in the cascade, there is a need of highly specific reporter molecules with the lowest possible signal background. Finally, methods with one or two reagent additions to each well of a nanoplate are preferred in a drug discovery process. Thus, a desirable screening technology should be a mix of: (1) the high sensitivity of reporter based methods; (2) the low false positive rate of second messengers methods; (3) the short assay times of second messengers methods that are transcription-free; (4) the stable signal of protein reporter based methods; (5) the minimal reagents additions or separations of assay products of homogeneous methods; (6) a robust signal with a high signal-to-noise ratio; (7) an amplifiable signal for reducing assay volumes while preserving a high signal-to-noise ratio and (8) a universal readout that could be used for the vast majority of human drugable genome.
Cell with regulated exocytosis of preformed reporters could meet several of the above conditions and thus such cell based sensors could be of high utility in drug discovery and compound characterization. Endogenous beta-hexosaminidase has been the most widely used lysosomal reporter for degranulation but this protein is considered to be a low sensitivity reporter. For example, Tiberghien et al (Tiberghien et al. Journal of Immunological Methods 223_1999.63-75) developed a method in which promyelocytic HL-60 cells were differentiated and employed to set up a 96-well microplate methodology using filtration instead of centrifugation to collect the extracellular fluid together with beta-hexosaminidase as the cell-released enzyme that was enzymatically measured. This method uses non-professional cells that need to be differentiated to induce secretion, both the beta-hexosaminidase reporter and the chemoattractant receptor are endogenous and thus low expressed and all the above combination of factors result in a method that needs at least 250.000 cells per well for screening. Thus, the authors claim that the main advantage of their method is the use of filtration instead of centrifugation to collect the extracellular fluid.
In another assay, Naal R M et al. Biosens Bioelectron 2004 Nov. 1; 20(4):791-6. Naal et al have developed a direct degranulation assay to enable the use of RBL-2H3 mast cells as a biosensor for screening chemical libraries for drug discovery and environmental toxicity evaluation based on the release of endogenous beta-hexosaminidase into the extracellular milieu in a single step. The authors anticipate the use of such method for detecting hapten-IgE interactions and for screening pharmacologic inhibitors of syk tyrosine kinase activity critical for degranulation. Those authors also use endogenous beta-hexosaminidase as reporter and only use the method for detecting hapten-IgE interactions and for screening of pharmacologic inhibitors of tyrosine kinases that participate in degranulation. In addition in this method only adherent cells are used and thus a washing step of each well is needed to eliminate background due to both basal beta-hexosaminidase activity accumulated during the 16 to 24 hours of cell culture before assay and due to beta-hexosaminidase activity normally present in bovine serum used in cell culture media. This washing step of individual wells limits throughput, increases costs and when done in a HTS environment with automatic pipeting robots the signal to background of assays is reduced due to residual volume in the wells containing beta-hexosaminidase activity. Finally as beta hexosaminidase enzyme is expressed by most hemopoyetic cell lines with professional regulated exocytosis, this enzyme allows only the development of monoplex assays and not multiplex assays.
In a third method, Graminski, G F et al (see Graminski G F et al J. Biol. Chem. (1993),268, 8, 5957-5964) have used pigment dispersion in frog melanophores mediated by receptors that activate protein kinase A or protein kinase C to rapidly evaluate chemicals for their effects on receptors that activate PKA or PLC via a functional assay that is used for investigations of ligand-receptor interactions and for massive drug screening. A major drawback of this method is that uses cells of non-mammalian origin for functional evaluation of receptor-ligand interaction and that colorimetric detection is of low sensitivity when compared with fluorescent or chemiluminescent methods.
Other methods have been developed to study intracellular trafficking and secretion of fusion proteins between a lysosomal targeted partner and a fluorescent protein, such as GFP. In a first method, El Meskini, R et al (see El Meskini R et al. Endocrinology 2001, 142-2, 864-873) have used preproneuropeptide Y fusions with GFP to explore routing of the chimeric proteins in AtT-20 cells, PC-12 cells, and primary pituitary cells to yield GFP storage in LDCVs that underwent stimulated release. At 2002, Rajotte (WO2004/016212) claimed he has developed a technology by fusing RMCP to GFP for detecting and quantifying degranulation but this method is only useful for measuring trafficking but not for quantification because of the low sensitivity of GFP released by the cells.
Other researchers have transfected GPCR into professional secretory cells like RBL-2H3 but endogenous beta-hexosaminidase have been always the reporter used to measure degranulation, only adherent cells has been used for assays and thus an additional washing step is needed to eliminate background thus compromising throughput and this enzyme only allows the development of monoplex assays. Also, promoters and conditions used for expression of surface receptors like GPCR into hemopoyetic cells such as RBL-2H3 need to be carefully optimized to find consistent results. For example, adenosine 3 receptor is considered a GPCR that does not degranulate by itself but potentiates degranulation induced by suboptimal amounts of IgE-allergen. Thus, current state of the art does not teach us how to develop a robust and sensitive sensor based on degranulation suitable for use in HTS.
Until the present invention, there have been no reports on the use of highly specific serine proteases like granzymes A, B, human chymase, proteinase 3 or neutrophil elastase as reporters stored in secretory lysosomes to develop hemopoyetic cell based sensors useful for drug discovery or to detect molecules for diagnostic. Current state of the art employs endogenous beta-hexosaminidase as a reporter by measuring the activity of this enzyme from at least 50.000 cells, a relative large number of cells (see Schwartz et al. J. Immunol. 123:1445-1450, 1979; and Dragonetti et al. J. Cell Sci. 1 13:3289-3298, 2000) or lysosomal enzymes fused to GFP to track the movement of secretory lysosomes is monitored in real time.
The present invention describes a highly sensitive hemopoyetic cell based sensor based on degranulation of protease reporters useful to test interactions between at least two molecules, with a high signal to background for robust detection, a fast kinetic, with minimal steps amenable for high throughput screening and using sensitive substrates of reporter enzymes for detection of secreted enzymes from a low number of cells to reduce costs. This cell based sensor could be used either in monoplex or multiplex. Multiplex assays have several advantages over monoplex assays for example: an increased throughput, cost reduction without compromising data quality or even improved data quality as every assay has as internal control the other assay made in the same well.